The long-term objective of the proposed research is to use morphological and cell biological techniques in conjunction with biochemical methods to analyze structurally and functionally the mechanism of insulin action, particularly at the receptormembrane level. These studies will use the adipocyte and isolated subcellular fractions, the cultured fibroblast and hepatocyte. A unique morphological marker, monomeric ferritin-insulin will allow these studies to be accomplished. Both transmission and scanning electron microscopy will be used. Correlative biochemical experiments will be performed where appropriate. The major areas to be studied are: a) Further characterization of the insulin receptor with emphasis on the possible microredistribution of receptors on the adipocytes after occupance and if there are major differences in handling of receptor bound ferritin-insulin by fibroblasts and hepatocytes versus adipocytes. The approaches will include computer quantitative reanalysis of ferritin-insulin distribution and the effect of temperature, bifunctional crosslinkers and sulfhydryl reagents on ferritin-insulin distribution. b) Determine the relationship of the groups of insulin receptors on adipocytes to the glucose-transport system. Sulfhydryl reagents and bifunctional crosslinker will be used to verify if some of the groups of insulin receptors form the new glucose transport sites generated by insulin and what role disulfide bonds play in holding these groups together as well as the glucose transport glycoproteins. c) Characterize and determine the biological importance of uptake of ferritin-insulin into adipocyte lysosomes These studies will use chloroquine, temperature variations ad bifunctional crosslinkers to determine the route of uptake into the lysosome and the fate of the insulin receptor in this uptake process.